The objective of the research proposed is to improve our understanding of the mechanisms involved in the biochemistry and regulation of steroid hormone biosynthesis. The proposed studies focus on the cholesterol side chain cleavage reaction, the initial and rate-controlling step in the pathway. Recombinant DNA methods will be used to express human ferredoxin reductase and human cytochrome P450scc in E. coli. The mature forms of the enzymes produced in E. coli will be purified and their properties compared with those of the naturally occurring enzymes. Recombinant human ferredoxin has previously been expressed in E. coli, and this will provide all three enzymes needed for study of the cholesterol side chain cleavage reaction. Site-directed mutagenesis is proposed to produce modified forms of each protein for structural and functional studies. For both ferredoxin reductase and P450scc, amino acid replacements will be made to investigate the role of specific residues in binding and electron transfer interactions with human ferredoxin. For ferredoxin reductase, substitutions will be made to identify residues involved in binding the cofactor, FAD, and the substrate, NADPH. For P450scc, substitutions will be made to identify residues important in determining steroid binding selectivity and specificity of the sites of hydroxylation. In addition, efforts will be made to obtain crystals of the recombinant proteins suitable for X-ray crystallography. The ability to express steroid hydroxylase components in bacteria will make larger amounts of these proteins available for future studies. It will also serve to avoid reliance on human (or other animal) tissues as a source of these proteins and reduce associated costs and risk factors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK030109-09
Application #
3229260
Study Section
Biochemical Endocrinology Study Section (BCE)
Project Start
1981-07-01
Project End
1996-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
9
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Type
Schools of Medicine
DUNS #
161202122
City
Irvine
State
CA
Country
United States
Zip Code
92697
Brandt, M E; Vickery, L E (1997) Cooperativity and dimerization of recombinant human estrogen receptor hormone-binding domain. J Biol Chem 272:4843-9
Cupp-Vickery, J R; Vickery, L E (1997) Crystallization and preliminary X-ray crystallographic properties of Hsc20, a J-motif co-chaperone protein from Escherichia coli. Protein Sci 6:2028-30
Vickery, L E; Silberg, J J; Ta, D T (1997) Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli. Protein Sci 6:1047-56
Xia, B; Cheng, H; Skjeldal, L et al. (1995) Multinuclear magnetic resonance and mutagenesis studies of the histidine residues of human mitochondrial ferredoxin. Biochemistry 34:180-7
Adlercreutz, H; Bannwart, C; Wahala, K et al. (1993) Inhibition of human aromatase by mammalian lignans and isoflavonoid phytoestrogens. J Steroid Biochem Mol Biol 44:147-53
Brandt, M E; Vickery, L E (1993) Charge pair interactions stabilizing ferredoxin-ferredoxin reductase complexes. Identification by complementary site-specific mutations. J Biol Chem 268:17126-30
Brandt, M E; Vickery, L E (1992) Expression and characterization of human mitochondrial ferredoxin reductase in Escherichia coli. Arch Biochem Biophys 294:735-40
Vickery, L E (1991) Active site-directed inhibitors of steroid hydroxylase cytochromes P450. Methods Enzymol 206:548-58
Brandt, M E; Gabrik, A H; Vickery, L E (1991) A vector for directional cloning and expression of polymerase chain reaction products in Escherichia coli. Gene 97:113-7
Coghlan, V M; Vickery, L E (1989) Expression of human ferredoxin and assembly of the [2Fe-2S] center in Escherichia coli. Proc Natl Acad Sci U S A 86:835-9